Plakortinic acids A and B: cytotoxic cycloperoxides with a unique bicyclo[4.2.0]octene unit from sponges of the genera Plakortis and Xestospongia

ABSTRACT

Plakortinic acids A (2) and B (3), two polyketide endoperoxides having an unprecedented bicyclo[4.2.0]octene unit, were isolated as minor constituents from the sponge-sponge symbiotic association  Plakortis halichondrioides - Xestospongia deweerdtae  from Puerto Rico, along with the known epiplakinic acid F (1). The molecular structures of 2 and 3 were determined mainly on the basis of NMR spectroscopy. Due to the structural similarities, 2 and 3 are thought to be biosynthetically related to 1. Biological screening for cytotoxic activity against two human tumor cell lines revealed that these novel metabolites are very active at low to sub-micro molar concentration.

GOVERNMENT INTEREST

This invention was made with U.S. Government support under grant number:GM086271 awarded by The National Institutes of Health (NIH). Thegovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

As part of our continuing efforts to identify new bioactive compoundsfrom Caribbean marine sponges, we re-investigated chemically thesymbiotic two-sponge association Plakortis halichondrioides-Xestospongiadeweerdtae collected in Mona Island off the west coast of Puerto Rico.Marine sponges within the genera Plakortis and Plakinastrella representan amazing source of cyclic peroxide-containing natural products, whichin addition to their interesting biological activity, display a diversearray of molecular architectures. Interestingly, many sponges of thePlakinidae family, as well as other marine animals, often contain aplethora of straight- and branched-chain 1,2-dioxolane carboxylic acidsof varying length that often incorporate multiple double bonds, terminalphenyl groups and, albeit rarely, cyclooctatriene rings (FIG. 3a ). Thisobservation entails that conjugated linear polyenes are ubiquitous inmany marine species.

Indeed, previous research has demonstrated that from relatively simple(E,E,E,E)-tetraene precursors, structurally diverse scaffolds such asthe bicyclo[4.2.0]octadiene core can be obtained through various modesof thermal and photochemical reactions. Thus, a suitable polyene couldbe encouraged by enzymes to undergo selective E/Z double bondisomerizations resulting in the necessary geometry for cascadeelectrocyclizations to ensue (FIG. 3b ). Bicyclo[4.2.0]octane-basednatural products with different substitution patterns have been reportedfrom various sources, including plants, saccoglossan mollusks,Streptomyces, and marine-derived fungi as shown in FIG. 2a -FIG. 2c .Although polyene precursors could give rise, among several complexskeletons, to compounds based on a bicyclo[4.2.0]octadiene framework,thus far this type of natural products have yet to be isolated from anyPlakortis or Plakinastrella species.

SUMMARY OF THE INVENTION

The present invention discloses the isolation and structure elucidationof plakortinic acids A (2) and B (3), the first two members of a newchemical series having an unprecedented bicyclo[4.2.0]octene backbone.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of the invention will become apparentfrom the following detailed description taken in conjunction with theaccompanying figures showing illustrative embodiments of the invention,in which:

FIG. 1 shows an underwater photograph of the sponge consortium Plakortishalichondrioides-Xestospongia deweerdtae.

FIGS. 2a-2c show bicyclo[4.2.0]octane-based natural products of theprior art.

FIG. 3a shows previously reported marine natural products arising from aplausible biosynthetic pathway involving a polyene precursor.

FIG. 3b shows biogenetic proposals for the formation of the phenyl andbicyclo[4.2.0]octadiene motifs from a linear polyene precursor.

FIG. 4 shows isolated polyketide-derived metabolites plakortinic acids A(2) and B (3) having a bicyclo[4.2.0]octene backbone, according to thepresent invention.

FIG. 5 shows a plausible biogenetic pathway of plakortinic acids A (2)and B (3) from epiplakinic acid F (1).

FIG. 6 shows ¹H NMR spectrum (CDCl₃, 500 MHz) of plakortinic acid A (2),according to the present invention.

FIG. 7 shows ¹³C NMR spectrum (CDCl₃, 125 MHz) of plakortinic acid A(2), according to the present invention.

FIG. 8 shows DEPT-135 spectrum (CDCl₃, 125 MHz) of plakortinic acid A(2), according to the present invention.

FIG. 9 shows HSQC spectrum (in CDCl₃) of plakortinic acid A (2),according to the present invention.

FIG. 10 shows HMBC spectrum (in CDCl₃) of plakortinic acid A (2),according to the present invention.

FIG. 11 shows expansion of the ¹H NMR spectrum (500 MHz) of plakortinicacid A (2) in Bz-d₆ (high-field region), according to the presentinvention.

FIG. 12 shows expansion of the ¹³C NMR spectrum (125 MHz) of plakinicacid A (2) in CDCl₃, according to the present invention.

FIG. 13 shows expansion of the ¹³C NMR spectrum (125 MHz) of plakinicacid A (2) in CDCl₃, according to the present invention.

FIG. 14 shows ¹H NMR spectrum (CDCl₃, 500 MHz) of plakortinic acid Bmethyl ester (4), according to the present invention.

FIG. 15 shows a low field region of the ¹H NMR spectrum (CDCl₃, 500 MHz)of compound (4), according to the present invention.

FIG. 16 shows a high field region of the ¹H NMR spectrum (CDCl₃, 500MHz) of compound (4), according to the present invention.

FIG. 17 shows another high field region of the ¹H NMR spectrum (CDCl₃,500 MHz) of compound (4), according to the present invention.

FIG. 18 shows ¹³C NMR and DEPTQ spectra (CDCl₃, 125 MHz) of plakortinicacid B methyl ester (4), according to the present invention.

FIG. 19 shows an expanded view of the ¹³C NMR and DEPTQ spectra (CDCl₃,125 MHz) of compound (4), wherein the signals highlighted are due to theminor component of the inseparable mixture of diastereomers, accordingto the present invention.

FIG. 20 shows HSQC spectrum (in CDCl₃) of plakortinic acid B methylester (4), according to the present invention.

FIG. 21 shows HMBC spectrum (in CDCl₃) of plakortinic acid B methylester (4), according to the present invention.

FIG. 22 shows suggested rationales for the NMR resonances that areobserved in pairs in compound (2) and (4), according to the presentinvention.

FIG. 23 shows a plot of cytotoxicity of plakortinic acid A (2),according to the present invention.

Throughout the figures, the same reference numbers and characters,unless otherwise stated, are used to denote like elements, components,portions or features of the illustrated embodiments. The subjectinvention will be described in detail in conjunction with theaccompanying figures, in view of the illustrative embodiments.

DETAILED DESCRIPTION OF THE INVENTION

In general terms, the compounds were obtained by cutting the spongespecimens into small blocks and blending with MeOH—CHCl₃. Afterfiltration, the extract was concentrated to yield a gum that wassuspended in H₂O and extracted with n-hexane. After concentration, aportion of the oil obtained was chromatographed with n-hexane-acetone.Fractionation and purification of active components guided by ourcytotoxicity assay resulted in the isolation of two novel, highlycytotoxic polyketide derived metabolites, plakortinic acid A (2, 8.0 mg,0.01% yield), and its counterpart plakortinic acid B (3), along withknown epiplakinic acid F (1, 170 mg, 0.21% yield), which had beenisolated before from this sponge genus. Treatment of an aliquot of 3with CH₂N₂ followed by successive CC and RP-HPLC yielded the methylester of plakortinic acid B (4, 12.0 mg, 0.02% yield), which wassuitable for structure elucidation work. FIG. 4 shows the isolatedpolyketide-derived metabolites plakortinic acids A (2) and B (3) havinga bicyclo[4.2.0]octene backbone. Although the presence of a majorproduct was evident in the NMR spectra of 4, the existence of minoramounts (<25%) of another stereoisomer was detected as shown in FIG.14-FIG. 19.

Experimental Section General Experimental Procedures

Optical rotations were obtained with an Autopol IV automaticpolarimeter. Infrared spectra were obtained with a Nicolet Magna FT-IR750 spectrometer. 1D- and 2D-NMR spectra were recorded with a BrukerDRX-500 FTNMR spectrometer. Mass spectrometric data were generated atthe Mass Spectrometry Laboratory of the University of Illinois atUrbana-Champaign. Column chromatography (CC) was performed using silicagel (35-75 mesh). TLC analysis was carried out using glass pre-coatedsilica gel plates, and the spots were visualized by exposure to I₂vapor. All solvents used were either spectral grade or distilled fromglass prior to use. Commercially available Diazald® and dimolybdenumtetraacetate were purchased from Sigma Aldrich Co.

Animal Material

All individuals, found along the ceiling of cave overhangs with a dropshape, were massively encrusting with irregular conulose surface. Mostspecimens collected measured up to 20 cm long and 5 cm thick. AllPlakortis halichondrioides colonies were overgrown with thethinly-encrusting sponge Xestospongia deweerdtae, which provides alavender pink crust over the olive green color of P. halichondrioides.However, specimens turned to a brownish color, producing a dark exudatewhen brought to the surface. Individuals were easily broken, with firmconsistency. Noticeable oscules along the surface were circular andmeasured 2.0-10.0 mm in diameter. The choanosome was compact with manycavities. Both choanosome and ectosome formed by high abundance of diodsthat were arranged homogenously and densely over the sponge body. Diodswere curved with sharp edges. Straight triods with sharp edges werehighly abundant. Nonetheless, many triods had rounded edges with a thickcenter. Triods and diods were variable in size. Minimum diod lengthvaried from 110 to 160 μm, and the maximum actine length of triod lengthvaried between 20 and 60 μm. The ectosome measured between 450 and 650μm thick. A high density of unusual cavities formed a mesh that ranperpendicular to the surface of the ectosome. An underwater photographof one of the sponge specimens is shown in FIG. 1.

Collection, Extraction, and Isolation

Fresh specimens of the sponge Plakortis halichondrioides (phylumPorifera; class Demospongiae; subclass Homoscleromorpha; orderHomosclerophorida; family Plakinidae) were collected by hand using scubaat depths of 90-100 ft off Mona Island, Puerto Rico. A voucher specimen(No. IM06-09) is stored at the Chemistry Department of the University ofPuerto Rico, Río Piedras Campus. The organism was frozen and lyophilizedprior to extraction. The dry specimens (395 g) were cut into smallpieces and blended in a mixture of CHCl₃-MeOH (1:1) (11×1 L). Afterfiltration, the crude extract was concentrated and stored under vacuumto yield a dark gum (100 g), which was suspended in H₂O (2 L) andextracted with n-hexane (3×2 L), CHCl₃ (3×2 L), and EtOAc (3×2 L).Concentration under reduced pressure yielded 16.4 g of the n-hexaneextract as a dark brown oil, a portion of which (3.7 g) waschromatographed over silica gel (130 g) using mixtures ofn-hexane-acetone of increasing polarity (0-100%). A total of 11fractions (I-XI) were generated on the basis of TLC and ¹H NMR analysis.Further purification of fraction II (1.3 g) by silica gel (20.0 g)column chromatography in 2% acetone-n-hexane afforded eightsub-fractions, denoted as A-H. Purification of fraction B (659.1 mg) bysilica gel (13.0 g) CC using CHCl₃ 100% as eluent afforded a mixtureenriched with plakortinic acid B (3) (22 mg, 0.04% yield). The morepolar fraction H (215 mg) was subjected to CC using reverse-phasedsilica gel (5 g) and eluted with a MeOH—H₂O gradient (6:4; 7:3; 8:2;9:1; 1:0) to yield 7 sub-fractions denoted as H1-H7. Subfraction H4 (30mg) was purified through a short plug of silica gel (0.8 g) with aCHCl₃-MeOH gradient (10:0; 9.5:0.5; 9:1) to afford plakortinic acid A(2) (8.0 mg, 0.01% yield). Purification of subfraction II (H) (659.1 mg)by silica gel (13.0 g) CC using CHCl₃ as eluent afforded the knownepiplakinic acid F (1) (170 mg, 0.21% yield).

Methylation of Plakortinic Acid B (3)

To a solution of impure compound 3 (22 mg, 0.052 mmol) in CHCl₃ (8 mL)was added a solution of diazomethane in ether (10 mL), and the resultingmixture was stirred at 25° C. for 2 h. The oily residue obtained afterconcentration was chromatographed using a short plug of silica gel (1.0g) and a mixture of n-hexane-acetone (95:5) to yield three fractions(I-III). Fraction II (15 mg) was submitted to reversed-phased HPLCchromatography (column: RP Analytical Hypersil 5μ C₁₈ 250×4.6 mm) usingisocratic elution with MeCN—H₂O (85:15, flow rate: 0.80 mL/min). AfterHPLC purification, four sub-fractions were obtained (IIa-d). SubfractionIIc yielded compound 4 (12.0 mg, 53% yield) as an inseparable mixture in3:1 ratio based on ¹H NMR integration.

The present invention will be explained in conjunction with the NMRspectra shown in FIG. 6-21. Plakortinic acid A (2) was optically activeand its molecular formula was determined as C₂₄H₄₀O₆ by HRESIMS (m/z447.2726, [M+Na]⁺, Δ +0.3 mDa) requiring 5 sites of unsaturation.Plakortinic acid A (2): colorless oil; [α]_(D) ²⁰+28.5° (c 1.3, CHCl₃);IR (film) u_(max) 3391, 2930, 2855, 1714, 1455, 1376, 1220, 1061 cm⁻¹;HRESIMS m/z [M+Na]⁺447.2726 (calcd for C₂₄H₄₀O₆Na, 447.2723). Thepresence of hydroxyl and carboxylic acid groups was implied from thebroad stretch at 3391 cm⁻¹ and sharp band at 1714 cm⁻¹, respectively.The ¹C NMR spectra (Table 1 below) showed 24 resolved signals that,together with the ¹³C DEPT-135 and HSQC NMR data, were assigned as4×CH₃, 9×CH₂, 7×CH, and 4×C; thus, compound 2 had to have 3 OH groups.The ¹H NMR spectrum showed one olefinic proton (δ_(H) 5.56, d, J=5.8Hz), two oxymethines (δ_(H) 4.11, dd, J=5.8, 3.3 Hz and 3.93, dd, J=9.7,3.3 Hz), three methyl singlets (δ_(H) 1.65, 1.46, and 1.29), and onemethyl triplet (δ_(H) 0.91, t, J=7.5 Hz). Some of these proton signalsand those later ascribed to H₂-2 and H₂-4 were consistent with a1,2-dioxolane bearing two methyl groups at C-3 and C-5 and an aceticgroup at C-3. All of the C—H correlations within 2 were established froma ¹H-¹C HSQC experiment. HMBC cross-peaks of H₂-2 with C-1, C-3, andC-22 and of H₂-4 with C-3, C-5, C-6, C-22, and C-23, confirmed that 2contained the same free 1,2-dioxolane carboxylic acid as epiplakinicacid F (1). ¹H-¹H COSY correlations ofH-16-H-15-H-14-H-13-H-12-H-19-H-18-H-13, along with the key HMBCcorrelations of H-18 (δ_(H) 2.34, t, J=8.4 Hz) with C-13, C-14, C-16,C-17, C-19, C-20, and C-24 and of H-13 (δ_(H) 2.41) with C-12, C-14, andC-18 led us to a bicyclo[4.2.0]octene ring system for plakortinic acid A(2). COSY, HMQC, and HMBC data routinely established the spin systemsfrom H₂-11 through H-16, H-18 through H₃-21, including H-13 and H-18 andthose of H-12/H19. These correlations also helped us establishunequivocally the locus of the 1,2-diol array at C-14/C-15. Furthermore,the HMBC correlation of H₃-21 (δ_(H) 0.91, t, J=7.5 Hz) with C-19 andC-20 allowed us to attach an ethyl group to C-19. Based on theseobservations, we concluded that the remaining NMR signals had to bethose of a straight alkyl side chain (C6 through C11) connecting thebicyclo[4.2.0]octene and cycloperoxide ring units.

TABLE 1 NMR Spectroscopic Data for 2 in CDCl₃ position δ_(C) ^(a) δ_(H)^(b) (J in Hz) HMBC (H→C#)  1 174.5, C  2a 43.9, CH₂ 2.76, d (14.8) 1,3, 4, 22  2b 2.72, d (14.8) 1, 3, 4, 22  3 83.9, C  4a 55.7^(c), CH₂2.25, d (12.5)^(c) 2, 3, 5, 6, 7, 22, 23  4b 2.44, d (12.5)^(c) 2, 3, 5,6, 22, 23  5 86.6^(c), C  6ab 39.5, CH₂ 1.70, m; 1.53, m 7  7ab24.3^(c), CH₂ 1.38, m; 1.30, m  8 29.8c, CH₂ 1.30, m  9 29.7, CH₂ 1.30,m 10ab 28.2^(c), CH₂ 1.39, m; 1.28, m 11ab 30.7, CH₂ 1.58, m; 1.38, m 9,12 12 39.8, CH 2.03, m 20 13 35.6^(c), CH 2.41, q (8.9 Hz) 12, 14, 18,19 14 68.9^(c), CH 3.93, dd (9.7, 12, 13 3.3) 15 67.8, CH 4.11, dd (5.8,13, 14, 16, 17 3.3) 16 121.3, CH 5.56, d (5.8) 14, 15, 18, 24 17 143.1,C 18 42.1, CH 2.34, t (8.4) 13, 14, 16, 17, 19, 20, 24 19 49.2, CH 1.66,m 12, 18, 21 20ab 28.8, CH₂ 1.58, m; 1.49, m 12, 18, 19, 21 21 11.9, CH₃0.91, t (7.5) 19, 20 22 23.8, CH₃ 1.46, s 2, 3, 4 23 23.2^(c), CH₃ 1.29,s 4, 5, 6, 7 24 21.3, CH₃ 1.65, s 16, 17, 18 ^(a)Recorded at 125 MHz.Multiplicities were obtained from a DEPT-135 experiment. ^(b)Recorded at500 MHz. ^(c)Two sets of signals with partial overlap)

The relative configurations of the stereocenters of the strained ringsystems in plakortinic acid A (2) were established on the basis ofcorrelations in the NOESY NMVR spectrum as well as throughinterpretation of NMVR coupling constant data (Table 1). The H₂-4 proton(δ_(H) 2.44) that is cis to the acetic acid side chain appears downfieldfrom the proton trans to the acetic acid. Thus, H-4b showed strongdipolar coupling to H₃-23. H-4a (δ_(H) 2.25) showed strong dipolarcouplings to H₃-22 and H₂-6. All of these are consistent with a transrelative stereochemistry for the 1,2-dioxolane carboxylic acid moiety of2. The assignments shown in 2 of C-3 and C-5 are not arbitrary. Rather,they are based on nearly identical NMR and [α]_(D) data plus the NOESYexperiments which showed correlations identical to those reported forthe co-isolated known compound 1. Cross-peaks of H-13 with H-12 andH-18, and of H-12 with H₃-21 placed these protons on the same face ofthe bicyclo ring system. Due to the near coincidence in chemical shiftsbetween H-13 and H-18 in CDCl₃, no consideration about their relativeorientation could be established on the basis of NOESY experiments.However, in Bz-d₆ greater dispersion in the chemical shifts made itpossible to detect an NOE cross-peak between these nuclei. While thoseof H-14 with H₂-11, H-15, and H-19 were used to place them on theopposite face. The conspicuous absence of NOE's between H-19, H-12, andH-18 confirmed their trans quasi-diaxial relationship. The couplingconstant for H-13 and H-14 (J=9.7 Hz) is in full agreement with theirtrans-diaxial orientation, whereas the small coupling constant betweenH-14 and H-15 (J=3.3 Hz) supports the cis geometry for the 1,2-diolarray in 2. Therefore, the relative configuration of plakortinic acid A(2) was determined to be 3S*, 5R*, 12S*, 13S*, 14R′, 15S*, 18R′, 19S*.An attempt to assign the absolute configuration of the 14,15-diol moietyin 2 using the dimolybdenum CD method was unsuccessful as the CDspectrum did not display a measurable Cotton effect.

Plakortinic acid B methyl ester (4) was obtained also as an opticallyactive substance. Plakortinic acid B methyl ester (4): colorless oil;[α]_(D) ²⁰=+12.6° (c 0.54, CHCl₃); IR (film) u_(max) 2932, 2854, 1738,1456, 1376, 1210, 1088, 1012, 715 cm⁻¹; HRESIMS m/z [M+H]+437.2910(calcd for C₂₅H₄₀O₆, 437.2903). Its HRESIMS indicated a [M+H]⁺ ion peakat m/z 437.2910, suggesting a molecular formula of C₂₅H₄₀O₆, from which6 degrees of unsaturation could be deduced. The ¹³C NMR and DEPT-135 NMRspectra (Table 2) exhibited 25 signals for 5 methyl, 9 methylene, 7methine, and 4 quaternary carbons. The 1D and 2D NMR spectra of 4revealed strong structural analogies with compound 2, suggesting thepresence of the same carbon skeleton. However, in place of acyclohex-3-ene-1,2-diol system, compound 4 was characterized by amethine linked to an oxygen atom (δ_(H) 4.63, H-14; δ_(c) 72.5, C-14),an oxygenated tertiary sp³ carbon at δ_(c) 77.3 (C-17), and a1,2-disubstituted double bond [δ_(H) 6.76 (H-15), 6.34 (H-16); δ_(c)132.6 (C-15), 135.8 (C-16)]. According to these data, a peroxide bridgeacross C-14/C-17 was suggested for compound 4. Conceivably, compound 3could be an artifact formed during work-up via 1,4-addition of O₂ to abicyclo[4.2.0]octadiene precursor. The remaining part of the moleculewas the same as 2. All proton and carbon resonances were attributed asreported in Table 2 by detailed NMR analysis (¹H-¹H COSY, HSQC, andHMBC).

In spite of their structural dissimilarities, epiplakinic acid F (1) andplakortinic acid A (2) have [α]_(D) values of the same positive sign andsimilar magnitudes. Although this could be dismissed as a coincidence,it might also imply that plakortinic acid A actually consists of aninseparable mixture of two diastereomers, namely, 2 (3S*, 5R*, 12S*,13S*, 14R*, 15S*, 18R*, 19S*) and 2′ (3S*, 5R*, 12R*, 13R*, 14S*, 15R*,18S*, 19R*). In order to account for this interpretation, as shown inFIG. 22, we propose that the bicyclic backbone of plakortinic acid A (2)is formed spontaneously, i.e., without the assistance of enzymes, froman achiral tetraene precursor through 8π-6π electrocyclization cascadesas racemates (note: natural products whose biosynthesis involves an8π-6π electrocyclization cascade are typically isolated as racemates).Additional reactions (hereon assisted by enzymes) would then ensueleading to the 1,2-dioxolane unit. We surmise that the specific rotationof the inferred 1:1 mixture of diastereomers (2 and 2′) is setexclusively by the dominant influence of a chiral 1,2-dioxolane ofwell-defined configuration (3S,5R), and that any contributions to[α]_(D) arising from the bicyclo[4.2.0]octene systems cancel out. Thishypothesis is supported by the fact that, although compound 2consistently behaved as a pure compound during multiple chromatographicanalyses (TLC, CC, HPLC), a handful of NMR signals (in particular thoseascribable to the 1,2-dioxolane array) were observed as pairs. Thisphenomenon appears to heighten in Bz-d₆ solution. This hypothesis couldalso explain why we were not able to establish the absoluteconfiguration of the 14,15-diol array in plakortinic acid A using the insitu dimolybdenum CD method developed by Frelek. On the basis of thisrationale, we also propose that plakortinic acid B was isolated as aninseparable 3:1 mixture of isomers 3 and 3′ (the assignment ofstructures is arbitrary).

As a second rationale, as mentioned earlier, in the case of plakortinicacid A, the initial characterization by ¹H and ¹³C NMR was complicatedbecause of the duplication of some of the proton and carbon signals.Conceivably, rotation around the straight alkyl chain gives rise to (atleast) two quickly interchanging rotational isomers with subtledifferent chemical shift values in a ratio of approximately 1:1.Unfortunately, we did not try to confirm this phenomenon by running theexperiments in DMSO-d₆ (with the hope of observing the coalescence ofthe duplicating peaks). Rotational isomerization is a reasonablerationale given the inherent conformational flexibility introduced bythe —(CH₂)₆— bridge of 2 and 3.

TABLE 2 NMR Spectroscopic Data for 4 in CDCl₃ position δ_(c) ^(a) δ_(H)^(b) (J in Hz) HMBC (H→C#)  1 171.1 C  2a 44.0, CH₂ 2.76, d (14.5) 1, 3,4, 22  2b 2.65, d (14.5) 1, 3, 4, 22  3 83.9, C  4a 55.4, CH₂ 2.22, d(12.6) 2, 3, 5, 6, 22  4b 2.47, d (12.6) 2, 3, 5, 6, 23  5 86.4, C  6ab39.6, CH₂ 1.69 m; 1.52, m  7 24.4, CH₂ 1.32, m  8 29.6, CH₂ 1.25, m  929.6, CH₂ 1.25, m 10 28.3, CH₂ 1.13, m 11 30.7, CH₂ 1.35, m 12 31.3, CH2.04, m 11, 13, 14 13 35.2, CH 3.04, ddd (9.2, 14, 18 9.0, 4.8) 14 72.5,CH 4.63, t (5.3) 13, 15, 16, 18 15 132.6, CH 6.76, dd (8.1, 13, 14, 176.2) 16 135.8, CH 6.34, d (8.1) 14, 17, 18, 24 17 77.3, C 18 42.5, CH2.30, dd (8.3, 13, 16, 17, 20, 24 5.8) 19 44.8, CH 1.52, m 12, 17, 2020ab 29.9, CH₂ 1.38, m; 1.28, m 21 21 11.9, CH₃ 0.81, t (7.3) 19, 20 2224.1, CH₃ 1.43, s 2, 3, 4 23 23.2, CH₃ 1.28, s 4, 5, 6 24 20.5, CH₃1.32, s 16, 17, 18 —OCH₃ 51.7, CH₃ 3.69, s 1 ^(a)Recorded at 125 MHz.Multiplicities were obtained from a DEPT-135 experiment. ^(b)Recorded at500 MHz

The relative stereochemistry of compound 4, suggested by comparison oftheir proton and carbon chemical shifts, was based mainly on NOESYexperiments. The expected cis geometry of the 13,18 junction wassuggested by a diagnostic NOE effect between the angular methines atthose sites. The peroxide bridge was α-oriented with respect to theplane of the bicyclo[4.2.0]octene by the downfield shift of H-13, whichin compound 4 resonated at δ 3.04 vs δ 2.41 of 2, suggesting that boththe peroxide unit and the angular methine at C-13 were on the same sideof the molecule. Additional NOE's, particularly H-16/H-19, H₂-11/H-15,H-12/H-13 and H-18/H₃-21 fixed the relative stereochemistry shown in 4.

Compounds 1 and 2 have common structural features, including almostcoincidental molecular formula and specific rotation. Except for abranching methyl group, both have identical 1,2-dioxolane rings andsame-length side chains. Considering these structural features, 1 islikely a putative biogenetic precursor of 2 and 3 as shown in FIG. 5.

Compounds 2 and 4 were found to be primarily responsible for thecytotoxicity of the crude extract of the sponge.

Cytotoxicity Assays

DU-145 human prostate cancer and A2058 melanoma cell lines were obtainedfrom ATCC. These cells were cultured in RPMI-1640 or DMEM mediumcontaining 10% fetal bovine serum (FBS), 100 units/mL penicillin, and100 μg/mL streptomycin. All cells were maintained in a 5% CO₂ atmosphereat 37° C. To determine the viability of the cells, Promega CellTiter 96aqueous nonradioactive cell proliferation assays (MTS) were performed asdescribed by the supplier (Promega; Madison, Wis.). Briefly, cells(5000/well) were seeded in 96-well plates and incubated overnight at 37°C. in 5% CO₂. Cells were treated for 48 h with each compound. Theconcentration used was 10 μM. Dimethyl sulfoxide (DMSO) was used as thevehicle control. IC₅₀ values of compounds were determined in adose-dependent manner (0.1, 0.5, 1, 5, 10, 20, and 50 μM). Cellviability was determined by tetrazolium conversion to its formazan dye,and absorbance of formazan was measured at 490 nm using an automatedELISA plate reader. The production of formazan dye was directlyproportional to the number of living cells. Each experiment was done inquadruplicate in the absence of a positive control. FIG. 23 shows thebehavior of plakortinic acid A (2) in our MTS assay versus A2058melanoma and DU-145 prostate cancer cells. The cells were significantlymore sensitive to compound 2 (IC₅₀'s 0.3 and 0.5 μM, respectively) than4 (IC₅₀'s 4.7 and 5.9 μM, respectively).

Although the present invention has been described herein with referenceto the foregoing exemplary embodiment, this embodiment does not serve tolimit the scope of the present invention. Accordingly, those skilled inthe art to which the present invention pertains will appreciate thatvarious modifications are possible, without departing from the technicalspirit of the present invention.

The invention claimed is:
 1. An isolated compound consisting of:

wherein R═ CH₃.